The “sugar chain” is a generic term of a molecule in which monosaccharides and derivatives thereof bond to each other to form a chain structure via glycoside linkages. The monosaccharide includes glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid and the like.
Sugar chains are very rich in diversity and involved in various functions of naturally occurring organisms. Sugar chains, which frequently occur in vivo as complex carbohydrate bound to protein or lipid, are one of major constituents of a living body. It has been revealed that sugar chains in vivo are deeply involved in intercellular signal transduction, regulation of functions and interactions of proteins, and the like.
As a biological macromolecule containing sugar chains, there may be mentioned, for example, cell wall proteoglycans in plant cells, which contribute to stabilization of cells, glycolipids, which affect differentiation, proliferation, adhesion, movement or other behaviors of cells, glycoproteins, which are involved in intercellular interaction, cell recognition, and the like. There have been gradually unveiled mechanisms in which sugar chains in these macromolecules control sophisticated and accurate biological reactions through acting for, replacing, aiding, enhancing, regulating or inhibiting their functions each other. Further, if roles of sugar chains in differentiated proliferation of cells, cell adhesion, immunity and canceration of cells are clarified, new development can be expected through close connection of glycoengineering with medical science, cell engineering or organ engineering.
As an example of such progress, there may be mentioned that study has been actively pursued on occurrence of diseases caused by malfunction of sugar chains in cell surfaces or abnormal sugar chain-receptor interactions, roles of sugar chains in virus infections such as AIDS, and the like. Furthermore, study on involvement of sugar chains in cell-cell interactions or cell-matrix has become more important for understanding biological reactions (see, for example, Non-patent document 1).
For analysis in such study, there have been developed technologies for structural analysis of sugar chains. These technologies are combinations of steps such as liberation of sugar chains from complex carbohydrates, separation and purification of the sugar chains and labeling of the sugar chains. These steps are very complicated. In particular, the step of separation and purification, wherein only sugar chains are recovered from a sample contaminated with impurities, is very difficult and requires highly sophisticated skills.
For separation and purification of sugar chains, there have been used techniques such as ion exchange resins, reverse phase chromatography, activated carbon, and gel filtration chromatography. However, since these techniques are not a method for specifically recognizing sugars, contamination by impurities (such as peptide and protein) may not be avoided, and in many cases recovery efficiency of sugar chain varies depending on its structure. Furthermore, when sugar chains are separated by chromatography with a high degree of accuracy, fluorescence labeling, such as pyridylamination, of the sugar chains is necessary, which requires a complicated operation. In order to analyze the fluorescence-labeled sugar chains, it is necessary to purify the labeled sugar chains by removing impurities such as unreacted 2-aminopyridine from the reaction solution after labeling.
In general, the impurities are removed by gel filtration with the use of the molecular weight difference between the labeled sugar chains and the impurities. However, this method is difficult to treat a large number of samples in short period because it uses many instruments and requires much time. Although removing the impurities by azeotropic distillation has been attempted as a simple method, it is difficult to sufficiently remove the impurities. In order to clarify the relationship between sugar chain structures and various diseases, it is required to investigate sugar chain structures of a large number of samples so that data can be statistically treated. In this case, use of complicated techniques like conventional methods would require huge amount of cost and time. Consequently, there has been demanded a means to separate and purify sugar chains by a simple operation.
Moreover, in analyzing sugar chains involved in various biological reactions, a biochip may be a powerful tool.
Here, the biochip is one of biochemical techniques detecting specific interactions by immobilizing biological substances such as nucleic acids, proteins and sugar chains or cells on a substrate and contacting the immobilized substances or the like (referred to as probe) with biological substances or other compounds (referred to as target), specialized for enabling high-throughput detection/analysis through performing a large number of interactions in parallel. There may be specifically mentioned a DNA chip (DNA microarray), which has been already widely used in the field of function analysis of genes, a protein chip, which is expected to be used in the future, and the like. The DNA chip is a chip wherein nucleic acids are immobilized on its substrate at a high density and the presence of their complementary sequences is detected through hybridization. The protein chip is a chip wherein proteins are immobilized and proteins that interact therewith are detected. A glycochip, wherein sugar chains are immobilized, is expected to greatly contribute to study on interactions between sugar chains and sugar chain receptors, between sugar chains and cells, and between sugar chains and viruses (for example, Non-patent documents 2 and 3). Furthermore, the glycochip is expected to be used as a diagnostic device for infectious diseases and diseases related with sugar chain abnormality. However, there has been no means to immobilize sugar chains on a substrate efficiently by a simple operation, and thus a method to solve this problem has been desired.
[Non-patent document 1] Cold Spring Harbor, Tousa Seibutsugaku, Maruzen Co., Ltd., 2003 (in Japanese); Essentials of Glycobiology, ed. by Ajit Varki, Cold Spring Harbor Laboratory Press, 2002.
[Non-patent document 2] Nature, 2003, vol. 421, pp. 219-220.
[Non-patent document 3] Current Opinion in Structural Biology, 2003, vol. 13, pp. 637-645.